The overall goals of the proposed Program Project are to identify the genetic determinants of SARS-CoV pathogenesis and virulence, and develop candidate live attenuated, killed, and recombinant virus vaccines that protect animal models from wild type virus challenge. The Program is based on extensive preliminary studies by an existing team with considerable expertise in coronavirus molecular genetics and replication, viral pathogenesis and vaccine design. Two overall hypotheses are central to the Program Project, both based on extensive molecular genetic and immunologic studies on coronavirus. We hypothesize that mutagenic inactivation of various genes and functional domains will reveal the molecular determinants of SARS pathogenesis and virulence following introduction of recombinant viruses into small animal models. These studies will allow for concurrent development of live attenuated viruses that could be used directly as vaccines, or as seed stocks for developing safer "killed" vaccines. This hypothesis is supported in the preliminary empirical studies demonstrating that specific mutations and genetic deletions can be introduced into the SARS-CoV genome and the successful rescue of viable viruses. Our second hypothesis is that existing virus vaccine platforms, either alone or in combination, will induce strong protective immune responses against the SARS-CoV. Thus, we anticipate that the project will 1) rapidly identify genetic determinants of SARS-CoV pathogenesis and virulence;2) produce a number of attenuated SARS viruses that serve as seed stocks for safer candidate live or killed vaccines and recombinant vaccines;3) evaluate the efficacy of early vaccine candidates in animals;and 4) select the best of these for further evaluation. Concordant with these pathogenesis and vaccine studies, the program uses SARS-CoV as a model to attack fundamental issues regarding the safety of live attenuated viruses that are germane to many important RNA and DNA viruses. Consequently, the program addresses a number of basic questions that are germane to elucidating the molecular mechanisms governing the evolution and the emergence of new coronaviruses in humans. Finally, the program develops a large number of novel reagents that will propel SARS-CoV research forward, including the development of an infectious cDNA, novel recombinant viruses, recombinant proteins, and antiserum directed against the SARS-CoV proteome.